| BackgroundScald injury is a kind of skin damage disease which is always caused by physical,chemical and other factors,and happens frequently in life.Different from other common traumas,scaldinjury is always accompanied by wound pain,tissue necrosis,susceptible to infection,scar after healing and other problems.Scald injury destroys the skin barrier firstly,and then leads to a series of stress reactions,and results in systemic pathophysiology,metabolism,immune and other complex changes,thus causing a series of complications such as sepsis,systemic inflammation response syndrome and multiple organ dysfunction syndrome.Clinically,some antibiotics and traditional Chinese medicines are used,but their curative effects are unsatisfactory.Wounds are easily to have repeated infection and ulceration,which makes patient miserable.Issues are also easy to form scar,which affects the appearance.Therefore,the development of new anti-scald drugs is necessary.Maggot,also known as Wuguchong in traditional Chinese medicine,belongs to Lepidoptera,is insect larvae of chrysomyiamegacephala,luciliasericata and other flies.Many ancient Chinese medicine books recorded the application of maggot in treating infectious surgical diseases.Clinical studies showed that maggot therapy has no effects on normal tissue without damage,and has anti-infection and healing promotion effects on wound in the process of debridement.Maggot protein,an active ingredient isolated from maggot has antibacterial activity and many other functions including anti-virus,improving immunity,scavenging oxygen free radicals,slowing down skin aging process and anti-cancer etc.But there is no report on the therapeutic effect of maggot protein on skin scalds.Therefore,the development of maggot protein preparation which is safe and effective has great significance for the treatment of skin scalds.Objectives1.To separate and extract the protein components in maggot,and evaluate the preliminary pharmacodynamics;2.To develop maggot protein gel preparation;3.To establish the quality standards for the maggot protein gel preparation;4.To study the pharmacodynamic effect of maggot protein gels in rat scald injury model.Methods1.Extraction of the maggot protein and evaluation of the preliminary pharmacodynamics: The worm eggs of luciliasericata were cultured by sterile feeds.To obtain the sterile maggots,polypides and eggs were both disinfected by “Double sterilization method”,and then bacteriological and virological examinations were carried out.The maggot protein was made through grinding,hearting,and centrifugation methods.The deep Ⅱ degree scald model was induced by hot water on the back of rats.Then,maggot protein(02.g per wound)was given once a day and that lastedfor 14 days.The healing state of the wound was observed,and the healing rate and healing time were recorded.The hydroxyproline content in the wound tissue was also measured.Different fragments of the maggot protein were separated by cross-linked dextran G-50 column according to the absorption at 280 nm,and molecular weights were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The minimum inhibitory concentration(MIC)of P6 was determined,and the antibacterial activity of P1-P7 was determined according to the MIC of P6.The effect of each component on hydroxyproline content was also determined.2.Preparation of maggot protein gel: The mixtures of common used gel bases such as carbomer-carbomer-940,sodium carboxymethyl cellulose,high-substituted hydroxypropylcellulose,hydroxypropylmethylcellulose with maggot extract were investigated,and the maggot protein gel prescription was initially identified.A standard of gel screening score was developed to investigate the maggot protein gel.The effects of various factors on the stability of gels were investigated by single factor experiment and orthogonal design experiment.The effects of various factors on the stability of gels were investigated by evaluating the amount of carbomer-940,propylene glycol and pH.Finally,three gels were scored according to the orthogonal experiment,and the best formulation of gel was confirmed.3.The quality standard of the maggot protein gel was established: The external properties,pH,granularity,sterility,content uniformity and protein content of three batches of maggot protein gel were determined according to relevant provisions in the "Chinese Pharmacopoeia,2010 edition".Related quality standards were made according to the experimental results.In addition,the stability of the maggot protein gel under centrifugal,cold and high heat conditions were measured.4.Pharmacodynamics study of maggot protein gel: The deep Ⅱ degree scald model was induced by hot water on the back of rats.Then,maggot protein gels(0.2g per wound)was given once a day and that lasted for 14 days.Changes in the wound surface were recorded.The blood and wound tissues were collected after the rats were sacrificed.The histopathological changes of wound tissues were observed.The content of indicators including hydroxyproline,malondialdehyde(MDA),superoxide dismutase(SOD),tumor necrosis factor-a(TNF-α)and interleukin-6(IL-6)in sample were measured by the kit.The expression of EGF and bFGF was detected by immunohistochemistry.Results1.Extraction of maggot proteins and its preliminary pharmacodynamics evaluation: The bacteriological and virological examination showed that there were no bacteria or virological infection of the maggot,indicating that maggot could be further applied.Maggot protein treatment significantly reduced the healing time,increased the healing rate,and promoted the production of HYP.7 components(P1-P7)were isolated according to the different UV absorption characteristic peaks.The MIC of P6 on Escherichia coli and Staphylococcus aureus was 50mg/ml.Under the same conditions,the diameter of the antibacterial ring of the P6 group was significantly larger than that of the control group and other components.The activity of improving the hydroxyproline content in scalded wounds was also better than that of the other components.Thus,the maggot protein P6 component was applied to the next experiment.2.Preparation of maggot protein gel: According to the traits of different matrix and fly larvae protein extracts,carbomer-940 was chosen to be the gel substrate.The dosage of maggot protein P6 component in gel prescription was 5g according to the experimental results ofMIC.According to the results of single factor experiments and orthogonal design experiments,P6 5g,carbomer-940 0.6g,propylene glycol 10 g,Tween-80 0.2g,phenoxybenzoate 0.1g was the best prescription.The pH was readjusted to 6.5 by the amount of triethanolamine.The validation results showed that the gel was stable.3.The quality standard of the maggot protein gel was established: The pH value should be between 6.50-6.65.The granularity should be below 180μm.No bacteria should be detected in the gel.The marked loading capacity was tentatively set to 20 g.The protein content determination method in maggot protein gel was established,and the results of linear relationship showed that the constant of linear relationship r = 0.9990,indicating that the absorbance and the mass concentration had linear relationship in the range of 40.4 ~ 404.0μg/ml.The RSD of 6 times measured absorbance was less than 2%,indicating the instrument precision was good.The RSD of 7 times measured absorbance between 30 min was less than 2%,indicating the solution had good stability in 30 min.The absorbance measurement results were put into a linear equation,and protein contents of six copies test solution were obtained,the RSD value was less than 2%,indicating method reproduction was good.The absorbance measurement results were put into a linear equation,and protein contents of nine copies test solution were obtained,sample recovery rate was 99.56%(RSD<2%),which meet the requirements of 98%-102%.Finally,the protein content in maggot protein gel should be between 4.41% and 4.87%.The results of the stability study showed that the maggot protein gel was stable under the centrifugal,cold and high heat environment.4.Pharmacodynamics study of maggot protein gel: After the administration of maggot protein gel,the results of morphological observation showed that maggot protein gel significantly reduced the inflammatory response,accelerated the scab process in the early stage of trauma,shortened the healing time,and improved the wound healing rate.Pathological examination also showed that the skin appendage and neonatal epithelial tissue were covered byin maggot protein gel group,and a large number of newborn capillaries and fibroblasts,collagen arranged neatly,less granulocyte infiltration were found.After treatment of maggot protein gel,the content of hydroxyproline,MDA,TNF-α and IL-6 were decreased,and the contents of SOD,EGF and bFGF wasere increased.ConclusionsThe maggot protein has antibacterial and healing promotion effects,and the activity of seven main components isolated from the maggot protein were are screened,and the component P6 showsed the strongest activity.The gel in which P6 component was is used as the main drug was is stable and the quality can could be controlled.The maggot protein gel hashad the effect of promoting the scald healing and its mechanism might be related to the promotion of collagen synthesis,anti-inflammatory and anti-oxidation.Through this study,we have an in-depth understanding of the overall efficacy of the larvae protein and its gelon scald,and it provids a solid foundation for the development of drugs in future and mechanisms study. |
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